Method and Apparatus for Applying Aggregating Sampling to Food Items

ABSTRACT

Certain aspects of the present disclosure relate to methods and apparatus for microbial sampling of foods. For example, a method may include providing at least one aggregating sampler at one or more sampling locations, and sampling a production lot of produce or other food items such as meat using the at least one aggregating sampler to create one or more samples that makes up a microbial sampling. Certain aspects of the present disclosure relate to methods and apparatus for microbial sampling of foods. For example, an apparatus, such as a microbial aggregating sampler, may include a covering having a microbial sampling material with a pocket formed in the covering to receive an appendage or a tool for handling of the covering.

CLAIM OF PRIORITY UNDER 35 U.S.C. § 119

The present Application for Patent claims benefit of U.S. ProvisionalPatent Application Ser. No. 62/656,164, filed Apr. 11, 2018, U.S.Provisional Patent Application Ser. No. 62/589,755, filed Nov. 22, 2017,and U.S. Provisional Patent Application Ser. No. 62/543,220, filed Aug.9, 2017, assigned to the assignee hereof and hereby expresslyincorporated by reference herein.

BACKGROUND Field of the Disclosure

The present disclosure relates generally to improving the food safety ofready-to-eat produce and other food items and providing processvalidation, and more particularly, to methods and apparatus formicrobial sampling of food items and other materials.

Description of Related Art

The microbial testing process has undergone tremendous change in recentyears. Traditional plating techniques for enumeration and detection havegiven way to faster and more specific antibody and molecular biologybased techniques. These newer techniques may not require time forcolonies to form but they may generally require enrichment culture tocollect enough of the target organism and remove interference.

In the ready-to-eat produce industry, millions of dollars are spentcollecting grab samples attempting to demonstrate the safety of productsin an effort to meet demands by customers for an ever increasing numbersof tests. These efforts may be technically and statistically flawed andmay not meet the expectations of assuring food safety. Particularly,grab samples are too small to represent the production lots of material.Production lots of material are too heterogeneous for grab sampling tobe descriptive of the production lot. Further, results arrive too slowlyto make decisions without sacrificing quality. Pathogens levels aregenerally so low that the occasional positive sample reflects thebackground that is always present rather than a deviation from the norm.The ready-to-eat produce industry may benefit from an effective assay ofcross contamination and cross contamination control. It may also benefitfrom an effective measure of process efficacy and deviation inprocessing. Increasing the effectiveness of raw material testing mayhelp improve food safety practice.

Thus, as the demand for microbial sampling continues to increase, thereexists a desire for further improvements in sampling techniques andtechnology. Preferably, these improvements should be applicable to otherrelated technologies and the methods and devices that employ thesetechnologies.

BRIEF SUMMARY

The systems, methods, and devices of the disclosure each have severalaspects, no single one of which is solely responsible for its desirableattributes. Without limiting the scope of this disclosure as expressedby the claims which follow, some features will now be discussed briefly.After considering this discussion, and particularly after reading thesection entitled “Detailed Description” one will understand how thefeatures of this disclosure provide advantages that include improvedmicrobial sampling of foods and other products.

Certain aspects provide a method for microbial sampling of foods andother products. The method generally includes gathering a microbialsampling from one or more items, extracting microorganisms from themicrobial sampling, concentrating the microorganisms, cleaning themicroorganisms, tallying the relative presence of the microorganisms andany potential pathogens, aggregating this information of a microorganismtally from the tallying of microorganisms into a microorganism report,confirming the microorganism tally, and reporting the microorganismreport of the microorganism tally.

Certain aspects provide a method for microbial sampling of foods. Themethod generally includes providing at least one aggregating sampler atone or more sampling locations, and sampling, using the at least oneaggregating sampler, a production lot of produce creating one or moresamples that makes up a microbial sampling.

Aspects generally include methods, apparatus, systems, computer readablemediums, and processing systems, as substantially described herein withreference to and as illustrated by the accompanying drawings.

To the accomplishment of the foregoing and related ends, the one or moreaspects comprise the features hereinafter fully described andparticularly pointed out in the claims. The following description andthe annexed drawings set forth in detail certain illustrative featuresof the one or more aspects. These features are indicative, however, ofbut a few of the various ways in which the principles of various aspectsmay be employed, and this description is intended to include all suchaspects and their equivalents.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the manner in which the above-recited features of the presentdisclosure can be understood in detail, a more particular description,briefly summarized above, may be had by reference to aspects, some ofwhich are illustrated in the appended drawings. It is to be noted,however, that the appended drawings illustrate only certain typicalaspects of this disclosure and are therefore not to be consideredlimiting of its scope, for the description may admit to other equallyeffective aspects.

FIG. 1 illustrates example operations for microbial sampling, inaccordance with aspects of the present disclosure.

FIG. 2 illustrates example operations for applying aggregating sampling,in accordance with aspects of the present disclosure.

FIG. 3 illustrates a Table 1 that includes example treatments andresults for meat sampling, in accordance with aspects of the presentdisclosure.

FIG. 4 illustrates a Table 2 that includes example treatments andresults for leafy sampling, in accordance with aspects of the presentdisclosure.

FIG. 5 illustrates an aggregated sampler that includes a pocket inaccordance with aspects of the present disclosure.

FIG. 6 illustrates an aggregated sampler that includes a pocket thatextends through a covering in accordance with aspects of the presentdisclosure.

FIG. 7 illustrates an aggregated sampler with at least one convexsurface in accordance with aspects of the present disclosure.

FIG. 8 illustrates an aggregated sampler that includes at least oneconcave surface in accordance with aspects of the present disclosure.

FIG. 9 illustrates an aggregated sampler that includes a curved shape inaccordance with aspects of the present disclosure.

FIG. 10 illustrates an aggregated sampler that includes a hole or loopin accordance with aspects of the present disclosure.

FIG. 11 illustrates an aggregated sampler that is shaped as a mitten orglove for a hand in accordance with aspects of the present disclosure.

To facilitate understanding, identical reference numerals have beenused, where possible, to designate identical elements that are common tothe figures. It is contemplated that elements disclosed in one aspectmay be beneficially utilized on other aspects without specificrecitation.

DETAILED DESCRIPTION

Aspects of the present disclosure provide apparatus, methods, and/orsystems for automated and semi-automated microbial sampling of foods andother materials. Other materials can be as diverse as water or airstreams. More commonly, it will include kindred products of food such aspet food, medical materials or dietary supplements where microbialtesting is needed to confirm hygienic operation. Sampling can be activeas mediated by material flow or operator mediated by applying thesampler to a surface. Sampling can also be more passive and depend onpassive contact or gravity sedimentation.

The following description provides examples, and is not limiting of thescope, applicability, or examples set forth in the claims. Changes maybe made in the function and arrangement of elements discussed withoutdeparting from the scope of the disclosure. Various examples may omit,substitute, or add various procedures or components as appropriate. Forinstance, the methods described may be performed in an order differentfrom that described, and various steps may be added, omitted, orcombined. Also, features described with respect to some examples may becombined in some other examples. For example, an apparatus may beimplemented or a method may be practiced using any number of the aspectsset forth herein. In addition, the scope of the disclosure is intendedto cover such an apparatus or method which is practiced using otherstructure, functionality, or structure and functionality in addition toor other than the various aspects of the disclosure set forth herein. Itshould be understood that any aspect of the disclosure disclosed hereinmay be embodied by one or more elements of a claim. The word “exemplary”is used herein to mean “serving as an example, instance, orillustration.” Any aspect described herein as “exemplary” is notnecessarily to be construed as preferred or advantageous over otheraspects.

In one embodiment, devices are assembled and linked to a rapid reportingsystem to provide a more representative sampling and faster analysis ofa food product or other material. In another embodiment, a similarsystem provides for robotically sampling a field crop and deliveringresults with a mobile laboratory. Both embodiments can include atwo-stage screening system for speed and for economy, but a conventionaltesting approach can be considered to take advantage of the samplingimprovements.

There has been little evolution in sampling and sample preparation forsubmission to these advanced and rapid analytical techniques. Onetechnique is to collect periodic samples or random grabs from a lot.This sample is extracted by homogenization or stomaching and then eithera portion analyzed directly when high populations are expected orenriched prior to analysis. Liquid samples and particularly watersamples can be filtered to allow analysis of larger samples. For specialpurposes but generally not for routine analysis liquid samples can beconcentrated by centrifugation to pellet microorganisms. There arestatistically based sampling plans as recommended by academics orinternational organizations, but these sampling plans are rarelypractical and mostly cost prohibitive. Such sampling plans in use basedon periodic samples or random grabs have a very low data density.Furthermore, these sampling plans intrinsically have sampling biases dueto lot geometry where portions of the lot are essentially not sampledand the heterogeneous nature of microorganism distribution.

Some microbial testing may assume that the test organisms are evenlydistributed allowing a grab sample to be representative of the whole.This flawed assumption only accounts for inhomogeneous distributions inaggregate and requires many samples to characterize the microbialpopulation of a lot. Test and release inspection based on grab samplesmay be flawed to the extent that grab sampling can inherently misssignificant contamination. Furthermore, the size the sample limits thedetection limit to levels that may be order of magnitude above thebackground rate and the level where risk becomes imminent. On the otherhand, microbial proliferation during enrichment culture occurs underconditions selected to favor the growth of the potential pathogens whichoften have no relationship with the commercial conditions of storage andtherefore generates extorted perceptions of the microbial growth risk.

One or more embodiments as disclosed herein may address these shortcomings of the current sampling and testing practice and provides asystem to generate more meaningful, real time or near real time, onsiteassessment of microbial contamination risk.

Example of Automated and Semi-Automated Microbial Sampling of Foods andOther Materials

In accordance with one or more aspects of embodiments disclosed herein,automated and semi-automated microbial sampling of foods and othermaterials is provided. For example, devices may be assembled and linkedto a rapid reporting system to provide a more representative samplingand faster analysis of a food product. In another case, a system mayprovide robotic sampling of a field crop and may deliver results with amobile laboratory. Both examples may include a two-stage screeningsystem that may provide speed and for economy. Although lessadvantageous, it is reasonable to take advantage of improved samplingwith conventional enrichment and detection systems.

In one or more cases, a method and system of microbial sampling includesproviding a sampling sheet, such as a MicroTally Sheet, which is thenused with a continuous sampling device (CSD) to collect biologicalagents. The CSD may change the sampling sheet automatically betweensamples. The sampling sheet is then used for analysis. Particularly,target bacteria may be removed from the sampling medium. The samples oftarget bacteria are cleaned up and concentrated using a treater and madesuitable for analysis. The treated sample is then analyzed usingmolecular or biochemical methods and target agents are detected withacceptable accuracy and sensitivity. A cloud based data reporting systemwith user appropriate dashboards to report actionable information andfacilitate timely decision making may also be provided.

For example, FIG. 1 illustrates specific operations 100 for microbialsampling, in accordance with aspects of the present disclosure.

Specifically, operations 100 begin, at block 102, with gathering amicrobial sampling from one or more food items which may include, forexample, produce, meat, and other food products or other materials. Atblock 104, operations 100 include extracting microorganisms from themicrobial sampling. At block 106, the operations 100 includeconcentrating the microorganisms. The operations 100, at block 108,include cleaning the microorganisms. Further, the operations 100include, at block 110, tallying the relative presence of themicroorganisms and any potential pathogens. The operations 100 alsoinclude, at block 112, aggregating this information of a microorganismtally from the tallying of microorganisms into a microorganism report.At block 114, the operations include confirming the microorganism tally.The operations may also include, at block 116, reporting themicroorganism report of the microorganism tally.

In one or more cases, sampling may include using a sampling sheet orswab that collects a sample and is stomached in a 300 micron partitionedbag with 200 mls of eluting buffer and stomached. Concentration andcleanup of the sample may include siphoning an eluting buffer andentrained organisms through proprietary sequential filter and thetargets may be deposited on a 0.22 micron PC filter. The targets areanalyzed from the filter which may include for example, DNA purified andqPCR being run for Index elements including pathogen intensity, Entericstatus, and/or positive control. Cloud based reporting based on, forexample Ignition and SQL database, may be provided with user appropriatedashboards to provide timely actionable information

In accordance with one or more cases, pathogens may be confirmed byresampling and enrichment procedures, by resampling and doing definitivepathogen tests, or by doing confirmation tests on the original DNAdepending on the regulatory guidance. In one or more cases a cassetteand cartridge system may be implemented to streamline the samplingprocess. Although not required, eliminating the use of a stomacher andpartitioned bag may be provided in one or more cases. A bindingcollector may be provided to replace the PC membrane filter which mayhelp streamline the sample delivery to the detector and potentiallyeliminate the need for DNA purification. Use of Ribosomal RNA may becomea new standard for one or more such cases. In one or more cases, adetector may use flow amplification and laboratory on a chip typetechnology to further reduce detection times and costs.

To best address all the short comings in current practice numerousimproved elements may come together. Taken individually each improvementaddresses some of the short comings and yields some advantages. Leapingall the way to a complete solution may beyond the sophistication of someindustry classes so intermediate steps are considered for each element.For the initial discussion, the elements under consideration includeSampling, Extraction, Concentration, Cleaning, Screening Detection,Second Stage Sampling of Suspected Lots, Reporting, and Information RollUp. Although not as desirable, a more conventional enrichment can beused instead of Concentration. After such enrichment, any number ofdetection systems can be used to detect the presence or absence of atarget organism. Each of these elements is discussed below.

Thus, the timeframe from sampling to information may be driven by theneeds of the business class but is not limited thereto. Short shelf-lifeproduct can justify greater speed. Valuable commodities such as meatproducts will want more testing to limit exposure when a problem isexpected or anticipated. These factors will impact the degree to which acomplete automated solution is implemented or conversely when a systemmore akin to current practices is used to gain some of the benefits ofimproved sampling.

Examples of Sampling

There are practical limits to the amount of product that can be sampledby conventional means. Without heroic efforts samples are limited tosmall fraction of a pound (generally 150 grams or less but some labs areroutinely testing as much as 300 grams) which lead to operating curvesfor c=0 acceptance that have an inflection at about 1 CFU/pound. The neteffect of such sampling and testing is the erroneous belief that theworst lots are detected when 1 positive is found in many hundreds orthousands of samples. Unfortunately, this testing is so far removed fromthe range of interest; it is little more than a random selection of lotsto be rejected.

A manual sheet based sampling can increase the effective sampling weight20 or 30 fold which is enough to move the operating curve about an orderof magnitude to the left to about 0.1 CFU/pound if the same c=0inspection criteria are used. Similarly, if a continuous sampling methodis used the effective sample can be increased 200 to 300 fold yieldingan additional order of magnitude in LOD to about 0.01 CFU/pound.

The surface area of an aggregating sampler affords advantages beyondmaterial sampling when greater sensitive is desired for surface andfluid flow sampling. Water and air stream sampling are two examples ofwhere flow sampling is advantageous. The surface area is also applicableto the sampling of surfaces where topical contamination is of concern.

The use of the sampler has the advantages of being nondestructive andcan yield executional efficiencies. However, the real advantage comeswhen this LOD is traded off for statistical process control with atwo-stage acceptance criterion where deviation from normal are detectedas opposed to randomly selecting lots for reallocation. This concept isdiscussed more fully below when this discussion returns to screeningdetection. This is an important distinction when the goal is to improvethe microbial safety of a product or material. This line of argumentalso permits the more rapid detection of many cells rather than waitingfor one cell to grow into many cells. With the recognition of the powerof the larger and effective sampling procedure, there may be a need toexpand the range of tooling to apply this technology to a broader arrayof products with alternative geometries and increased levels ofautomation. For example, the geometry can be altered to allow samplingof a powder flow through a pipe with a circular geometry where a pipesegment is exchanged between lots. The pipe section would either belined with sampling material or better include baffles maximizingproduct contact with the sampling surfaces. Alternatively, one canenvision vertical chutes below pocket fillers to sample product justprior to bagging in a form fill and seal machine.

A microbial sampler may be included, for example, non-woven fabric,various micro fiber materials, sponges, and/or any absorbent sheetmaterial. A non-woven polypropylene or polyethylene fabric are ofparticular note as these materials are allowed for food contact andtherefore have very low extractables which might otherwise contaminantthe material stream under examination.

Additionally, the utility of these sampling approaches can be increasedwith automation. A feed cartridge can be used to deliver multiple sheetsto the sampling location at one time. This cartridge would be placed onthe line after sanitation has completed all preparations protecting thedrive mechanisms from the harsh cleaning process.

Similarly, a magazine of cassettes can be loaded to collect individualpieces of sampling material. Both the cartridge and cassette areengineered to advance the sampling material when appropriate, (e.g. Whena lot is completed, when a tote is moved, etc.). The motive force toadvance sampling sheets can be provided by a motor, or supplied by amanual crank or handle, or by an operator depending on the specifics ofthe operation. Both cassette and cartridge may be designed to protectthe microbial integrity of the contents. The cartridges may preventmicrobial growth after sampling, external contamination, and crosscontamination between lots.

The sampling material can be mounted on an inert backing material tofacilitate the sanitary placement of sampling sheets. Alternatively,sheets of sampling material can be separated by short spaces of inertmaterial to ensure that used sampling materials do not contaminate othersheets.

The usual design parameters for process equipment may be applied tothese devices including, for example, an aggregating sampler. Forexample, heavy gauge 316 stainless is an appropriate material. When thesampling device is not in place, the location may be passive such as adead plate where product passes without damage or hindrance. It may besanitary design from the beginning such that it is easily cleaned.

In one case, for this automation to have maximal benefit, the cartridgesmay carry the information regarding the sample they contain. If thesample is used in a manual mode, an electronic transfer of thisinformation along with the sample is also advantageous to avoid humanerror and speed the flow of information. This transfer of informationcan occur through the cloud using barcodes, a database and locationinformation.

Another class of geometries is necessary to extend the power of thissampling to agricultural commodities in the field. Such sampling hasutility beyond testing for human pathogens in that it can be used fortesting for plant pathogens that can decrease the productivity of acrop. For example, early detection of mildew spores prompt early harvestof a spinach field. Detection of blight in a wheat or corn field mightprompt the use of a disease control measure on the affected field beforethe blight destroys the crop.

Depending on the field crop, an octopus tentacle configuration may bethe appropriate geometry where strands of the sampling material areslide across the surface of the crop. These strands can be fuzzy cordsor strips of material depending on what provide the greatest effectivecontact. These tentacles can be contacted to the crop by variousmechanisms including robots, tractors, hand carrying or drones. It ismost important that the altitude be held constant to allow contact whileminimizing damage to the crop. To increase the effectiveness of thesampling, it can be advantageous to wick moisture down these tentaclesor install vacuuming or sucking mechanism.

For crops that present a more uniform top surface such as baby greens orspinach, sheet materials can be more effective as new upturned leavesurfaces are missed. For these crops, air based sampling with suction orelectrostatics that increases microbial sampling efficiency presents aninteresting alternative.

For manual sampling, forming the sampling material into pockets, mittensor gloves can facilitate use. Ease of use will generate greatercompliance with the sampling protocol. In a manual mode, the duration ofcontact is a factor in determining the effectiveness of sampling.Typical durations are minutes. 2-5 minutes durations will work for mostapplications.

FIG. 2 illustrates example operations 200 for applying aggregatingsampling, in accordance with aspects of the present disclosure.Specifically, operations 200 begin, at block 202, with providing atleast one aggregating sampler at one or more sampling locations.Additionally, operations 200 include, at block 204, sampling, using theat least one aggregating sampler, a production lot of produce creatingone or more samples that makes up a microbial sampling. The one or moresamples may be configured to be processed to indicate if pathogens arepresent at no greater than a normal background. The one or more samplinglocations may include at least one of in a field, at harvest, just afterdumping or cutting, in a wash system, or after the wash system. In oneor more cases, additional operations may be include such as, forexample, assessing, using the aggregating sampler, a level of crosscontamination control to validate or verify a wash process.

In one or more cases, an aggregating sampler may be provided thatsufficiently samples a production lot of ready-to-eat produce to confirmthat pathogens are present at no greater than the normal background. Inone or more cases, an aggregating sampler may assess the level of crosscontamination control to validate or verify a wash process.

In one or more cases, an aggregating sampler and a sampling location ofthe aggregating sampler may be provided. Additionally, sampling by theaggregating sampler may be provided to generate one or more desiredsamples. Analysis of the one or more samples and interpretation of theanalysis results may also be provided. These elements can be practicedindividually or together to enhance food safety.

The aggregating sampler may include a collection surface and anapparatus for holding and positioning the collection surface such thatthe collection surface contacts product that is to be sampled formicro-organisms or other targets. In one or more cases, a surface with asampling efficiency that allows an increased effective sampling sizewhen a production lot of the product is sampled may be provided. Forexample, two hours of production of a leafy green product may be sampledwith such a device. During the two hours a large amount, for example10,000 to 30,000 pounds, of product will have crossed the samplingsurface. If the sampling device is at least 25% efficient as shown inbench scale studies, the effective sample size may be 2500 to 7500pounds. These sample sizes are enormous when compared to the few hundredgrams of a normal grab sample.

In one or more cases, there are a number of factors to consider inselecting or designing an aggregating sampler. Initially, a samplingsurface that is compatible with the product is provided. This typicallymeans that the sampling surface is a food grade material. For example,in one or more cases, a non-woven polyolefin cloth has proven to beeffective. Another factor may include a design that allows safe andrapid exchange of the sampling surface. Another factor that may beincluded relates to any apparatus left on the line being easily cleanedand may incorporate a sanitary design. For example, food grade stainlesssteel may be a material selected when implementing an aggregatingsampler.

For produce testing, there are a number of areas where aggregatingsampling may yield improvements in food safety. For example, somelocations include: 1) In the field; 2) At harvest; 3) Just after dumpingor cutting; 4) In the wash system; or 5) After the wash system. In eacharea, there are particular locations that may be selected for aggregatesampling, but these will vary with the configuration of the specificline and the product to be sampled. Not all areas will be appropriatefor all products. The selections may be guided by the desiredinformation. Currently, grab samples are often taken in all of theseareas but these grab samples are unable to represent the populationunder examination and such sampling is generally destructive.

According to an example, an aggregating sampler may be provide that canreplace the current practice of taking a grab sample in a field bycutting leaves. Particularly, the aggregating sampler can be configuredsuch that it can be carried by hand or mounted on a device designed fortraveling through the field. The collection surface for field samplingmay be divided to allow more conformity to the crop surface.

According to another example, at harvest, an aggregating sampler may beplaced on the harvester such that product is sampled during theharvesting process. This approach would allow pairing of harvestedproduct with specific information. Although not required, placement ofthe aggregating sampler may be just after any sorting is done in thefield. For example, if rocks are sorted from the product by densityclassification, these rocks need not cross the sampler surface.

In another example, just after dumping or cutting and before washing,the product may still have the flora found in the field. An aggregatingsampler placed early in the process can sample these organisms. Justafter cutting or chopping, the interior of some products will be exposedfor the first time allowing a more representative sample to be taken.

In a wash system, an aggregating sampler can collect a different type ofsample in accordance with one or more examples. This sampler may testthe cross contamination control of the wash system. The organismscollected will reflect the two most probable mechanisms of crosscontamination, water mediated cross contamination and product to productcross contamination. An aggregating sampler, placed in the flow of theconveyed product will be impinged by both the water and the product. Toavoid overly hindering product flow depending on the line design, thesampler can be placed at any angle from parallel to the product flow tocomplete perpendicular to product flow. The sampler can also be acomb-like device with multiple collecting probes among product and inthe wash flow. The angle of attachment may affect the balance betweenwater mediated cross contamination and product to product crosscontamination observed. In either case, this type of sampling may beused to validate cross contamination control and effectiveness of washsolution.

Sampling in the wash system is a case where the sampling surface may beactive on multiple surfaces, for example, on both sides or around in thecase of the comb like structure mentioned above. In one or more cases,it can prove advantageous to laminate two sheets together with animpermeable tie layer to increase the binding potential relative to thedetachment potential by avoiding flow through the sampling surface.Additionally, further advantages may be provided in other ways such asby increasing the thickness of the sampling material. In some cases,designing samplers in devices such as filter housing may be provided. Inother cases, placement on a sampling surface in an active area of thewash system may be provided for getting a full measure of the crosscontamination potential.

A sample may be taken after the wash system and will reflect a residualpopulation. In this area there are a number of specific locations thatcan be considered depending on the specifics of the line. For example,these specific locations include just prior to loading dryers, in aconveyer that might be used to lift the product for packaging, justbefore a pocket scale, or in the throat of a form fill and seal machine.This in-line continuous sampling may significantly increase samplingefficiency and provide more meaningful data than grab sampling offinished product testing.

With samples taken, attention may turn to the analysis andinterpretation of results as discussed herein. For example, in thespecific category of produce, there are specific opportunities to beconsidered that may be provided with the aggregated sampling using theaggregated sampler. The opportunities are afforded in part due to themore representative nature of the aggregated samples and the greatlyincrease numbers of organisms available in the samples relative to thetypical grab sample. These samples may be analyzed to give multiplechannels of data depending on the detector technology employed. In oneor more cases, the sample may be analyzed with metagenomics, allowingfor the whole population to be studied yielding a large and in somecases a maximum amount of data which can be mined in various ways togain knowledge and understanding of positive and negative deviations.This range of possibilities can be illustrated with a number of examplesbut are not limited thereto.

In one or more examples, the samples taken in the field, at harvest, orjust prior to washing can be used to assess the microbiological statusof the raw material. From a food safety perspective the focus heretoforehas been on the presence or absence of pathogens. Unfortunately, suchanalyzes based on grab samples are unable to truly answer the questionas to whether pathogens are present due to their lack of sensitivity.Generally, it is known that pathogens are present at very low numbers.These are ubiquitous organisms. A more appropriate question is whetherthese organisms are present at abnormal concentrations or without theusual competing organism.

In one or more examples, samples taken just prior to washing may becompared to samples taken in the wash system to directly measure crosscontamination using wild type flora. Water samples may tend to have verylow microbial loads in properly managed wash systems even when crosscontamination is occurring. The wild type flora may also be highlyvariable. However, by using aggregating samplers at both locations thenoise can be dampened and cross contamination measured. A number ofmetrics for cross contamination can be considered based on the ratio ofthe results for the samples from prewash to those from in the wash. Byusing more sophisticated analytical procedures one can overcome theflaws in such metrics as aerobic plate count (APC) which would includemany organisms that are not relevant to cross contamination control ofpathogens. For example, spore forming bacteria such as Bacillus will beunaffected by the wash and just cloud any metric of cross contaminationbased on APC. However, with more focused channels of data as afforded bymodern molecular techniques better information can be obtained. Anotheraspect of this tool that may be provided is that statistical processcontrol can be applied to detect deviations.

In one or more cases, samples from after a wash process may provideinformation about microbial populations on the finished product. Thesesamples may provide a much more accurate assessment of the pathogen riskof the product and better detection deviations. These samples can alsobe used to check for deviations in the normal flora.

In some cases, a ratio between the before wash samples and the afterwash samples may be used to assess the impact of the wash process. Theaggregating samplers may reduce the noise and as with the crosscontaminations metrics, these ratios can be handled with statisticalprocess control to look for deviations.

One or more of these examples may be delivered in almost real timebecause the aggregating samplers collected enough cells forconcentration and direct analysis without enrichment.

Examples of Sampling: Bench Scale Examination of Sampling Efficiency forRaw Beef

In accordance with one or more cases, an example of a bench scale studydemonstrates a sampling efficiency of 15-20% relative to stomaching. Italso shows that transfer is essentially instantaneous and that repeatedcontact collects more organisms. These observations confirm theintuitive assertion that continuous sampling will yield betterinformation than grab sampling.

For example, for one bench scale study experiment, purchased stew meatis inoculated by immersion in a mixed culture of generic E. coli at ˜105CFU/ml. E. coli is selected as benign and easy to enumerate. The stewmeat is allowed to rest at room temperature for 30 minutes to allowadherence. Then the six treatments listed in the table shown in FIG. 3may be executed in 5 replicates. All sampling cloths may be cut in half,12 in by 8 in, to reduce the sample requirement and facilitatedexecution of the experiment. The sampling cloth may be, for example, aMicroTally cloth but is not limited thereto. The surface areas of themeat cubes are measured directly. For the sampling cloth treatments,meat is arranged in a 10 cm by 10 cm block and the sampling cloth isapplied to the upper surface. Each mini sampling cloth is extracted in200 ml of phosphate buffered saline (PBS). As a control, cubes of stewmeat are stomached for 60 seconds in 200 mL of PBS. All stomached cubesare measured to estimate surface area. The counts are normalized forsurface area and averaged. This normalization provides an apple to applecomparison.

This bench scale study experiment has been implemented and the averageresults are tabulated and shown in Table 1 of FIG. 3. These wereanalyzed with a General Linear Model (GLM) model which indicates thattime of contact was not a factor. Multiple contacts yielded about theexpected increase and are truly additive.

A logical extrapolation of this exercise is to estimate the effectivesample size of a sampling. This may not truly be possible given thedifferences in geometry. However, in one or more use cases when usingone side only, a sampling cloth is about 6 times larger than those usedin the bench scale study experiment and the intended use is to samplealmost 2000 pounds of product. Thus, it is reasonable to assert that theeffective sample is expected to be 300 to 400 pounds. Larger scaleexperiments may be implemented that may further confirm this estimate.In summary, a benefit and advantage of the above method and apparatus ofsampling may include providing an improvement over traditional grabsamples for meat sampling by providing larger effective samples.

Examples of Sampling: Bench Scale Examination of Sampling Efficiency forLettuce

In accordance with one or more cases, an example of a bench scale studydemonstrates a sampling efficiency of about 30% relative to stomaching.It also shows that transfer is essentially instantaneous and thatrepeated contact collects more organisms. These observations confirm theintuitive assertion that continuous sampling will yield betterinformation than grab sampling for lettuce.

For example, in accordance with a bench scale study experiment,purchased lettuce is inoculated by immersion in a mixed culture ofgeneric E. coli at ˜105 CFU/ml. E. coli is selected as benign and easyto enumerate. The lettuce is allowed to rest at 4° C. for 30 minutes toallow adherence. This short time may explain the higher efficiencyobserved when compared to a meat study where adhesion may be faster.Then the six treatments listed in Table 2 shown in FIG. 4 may beexecuted in 5 replicates. All sampling cloths may be cut in half, 12 inby 8 in, to reduce the sample requirement and facilitated execution ofthe experiment. The sampling cloth may be, for example, a MicroTallycloth but is not limited thereto. The surface areas of the lettuceleaves may be measured directly. For the sampling cloth treatments,lettuce is arranged in a 10 cm by 10 cm block and the sampling clothapplied to the upper surface. Each mini sampling cloth is extracted in200 ml of PBS. As a control, lettuce may be stomached for 60 seconds in200 mL of PBS. All stomached leaves are measured to estimate surfacearea. The counts are normalized for surface area and averaged. Thisnormalization provides an apple to apple comparison.

This bench scale study experiment has been implemented and the averageresults are tabulated and shown in Table 2 of FIG. 4. These wereanalyzed with a GLM model which indicates that time of contact was not afactor. Multiple contacts yielded about the expected increase and aretruly additive.

A logical extrapolation of this exercise is to estimate the effectivesample size of a sampling. This may not truly be possible given thedifferences in geometry. However, in one or more use cases when usingone side only, a sampling cloth is about 6 times larger than those usedin the bench scale study experiment and the intended use is to samplealmost 2000 pounds of product. Thus, it is reasonable to assert that theeffective sample is expected to be 400 to 600 pounds. Larger scaleexperiments may be implemented that may further confirm this estimate.In summary, a benefit and advantage of the above method and apparatus ofsampling may include providing an improvement over traditional grabsamples for leafy sampling by providing larger effective samples.

In one or more cases, swabs and/or sheets, referred to generally as asheet below, may be used in accordance with one or more embodiments ofthe present disclosure. A sheet may include a microbial samplingmaterial, such as sterile woven and/or non-woven synthetic fabrics andnon-woven cloth for sampling and testing in the field of food safety.These sheets can be folded and curved to allow better conformation toproduct (e.g., food) streams that are being sampled or when used in amanual mode as driven by the product and the container. In some cases,configurations for sampling raw products or materials may include asampling device that moves across the stationary product effectivelyyielding the equivalent of a product stream when sampling needs to occurprior to harvest. In some cases, configurations may include tail shapedsheets similar to, for example, the tentacles of a jellyfish.

In one or more cases, configurations may be provided that may provideeasier use under some conditions. In one case, a microbial aggregatingsampler, may include a covering including a microbial sampling materialwith a pocket formed in the covering to receive an appendage or a toolfor handling of the covering.

In some cases, the covering may further include an attachment featureformed in the pocket to receive the tool. The attachment feature mayinclude at least one of a hole formed through the covering; a looppositioned within the pocket to receive an end of the tool therethrough; or a tab positioned within the pocket for an end of the tool toattach thereto.

In some cases, the covering may include a sheath formed in the pocket toreceive a digit of an appendage. In some cases, the pocket is formedthrough the covering such that the appendage or the tool for handlingthe covering extends through the covering. The covering may becompletely formed from the microbial sampling material. In some cases,the covering includes two sheets attached to each other to form thepocket. In some cases, the covering may include a single sheet foldedand attached to itself to form the pocket.

For example, a sheet may undergo folding and seaming to form a bag orpocket that can be worn as a mitten, glove, sock, or other covering, tofacilitate manual sampling. Such a covering may encase an appendage,such as a hand, to facilitate pushing and pulling of the sampler throughthe product to be sampled. In some cases, aggregating samplers may becreated that are more hand like configurations as mitts with thumbsand/or gloves with fingers, allowing the sampler to better conform tothe hand. Such configurations may allow easier use when the product ismore viscous or more prone to adhering to the sampler. A benefit to thisaggregating sampler with a pocket may include the ability of the samplerto, when working the sampler, increase or maximize product contact.Further, in some cases, the addition of one or more attachment features,such loops or tabs, to assist with controlling the product contact maybe included. These are representative examples and are not meant tolimit other configuration embodiments of an aggregating sampler.

In some cases, the aggregating sampler may be used in an automatedmachine setting to sample a product stream. The aggregating sampler mayinclude a number of modifications in accordance with one or more cases.In some cases, the aggregating sampler may include one or more bends andcurves that may improve the utility and facilitate use. In some cases,modifications may include, but are not limited to, forming a tube thatcan slide over one or more shafts for positioning in the product flow.This configuration may remove the need to slide the sampler within anyholding device. In some cases, adding tabs or holes can be providedwhich may allow positioning and facilitate attachment. In some cases,the active sampling surface can be attached to a support material or webthat has one or more of these features. One or more of these cases andmodifications may help facility contact with the product stream withminimal manual intervention.

FIG. 5 illustrates an aggregated sampler 500 that includes a pocket inaccordance with aspects of the present disclosure. As shown theaggregated sampler 500 includes a covering 504 made of a microbialsampling material. The covering is formed such that it includes a pocketformed in the covering to receive an appendage or tool 502 within thepocket for handling of the covering.

FIG. 6 illustrates an aggregated sampler 600 that includes a pocket thatextends or is formed through a covering 604 in accordance with aspectsof the present disclosure. The pocket is formed such that the appendageor tool 602 for handling the covering 604 extends through the covering.

FIG. 7 illustrates an aggregated sampler 700 with at least one convexsurface of the covering 704 in accordance with aspects of the presentdisclosure. The covering forms a pocket for a tool or appendage 702.FIG. 8 illustrates an aggregated sampler 800 that includes at least oneconcave surface of the covering 804 in accordance with aspects of thepresent disclosure. The covering 804 forms a pocket for a tool orappendage 802. FIG. 9 illustrates an aggregated sampler 900 thatincludes a curved shape covering 904 in accordance with aspects of thepresent disclosure. The covering 904 is formed to include a pocket for atool or appendage 902.

FIG. 10 illustrates an aggregated sampler 1000 that includes a hole orloop 1008 in accordance with aspects of the present disclosure. Thecovering 1004 may include the hole or loop 1008, such as positionedwithin the pocket of the covering 1004 and attached to an inner surfaceof the covering 1004. This hole or loop 1008 may be used by a hook 1006of a tool 1002 to attach to the covering 1004.

FIG. 11 illustrates an aggregated sampler 1100 that is shaped as amitten or glove for a hand 1102 in accordance with aspects of thepresent disclosure. As shown, the covering 1104 of the aggregatedsampler 1100 is formed to contour to a hand 1102. In particular, asheath is formed in the covering 1104 in this embodiment to receive athumb of the hand 1102, though one or more sheaths may be used toreceive any digit of the hand 1102. Further, though only a hand 1102 isshown as an example appendage in this embodiment, the present disclosureis not so limited, as other appendages (i.e., a foot) may becontemplated for other embodiments without departing from the scope ofthe present disclosure.

In accordance with one or more cases, the aggregated sampling sheet maybe hung into an active zone of a wash line with a cord or cable. Thisline may be attached in number of ways to the sampling material such as,for example, a grommet and clasp. In some cases, a ball and slide clampmay be used. In some cases, a punched hole may be used but may pull out.In other cases, various other clamps may be used. In one or more case,an optional float such as a fishing float with or without a weight maybe included that will add drag and bouncy which may improve surfaceexposure. This approach may not include a fixed appliance for holdingthe sampling material.

Examples of Extraction

The reference method for extracting microorganisms from grab samples ishomogenization by mixing or stomaching with an appropriate amount offluid, usually a buffer. The purpose of adding fluid is to neutralizeany antimicrobial or other properties of the sample that may beunfavorable for microbial growth such as low pH and suspendmicroorganism in liquid to facilitate downstream testing. However,adding fluid also dilutes the concentration of the microorganisms in thesample. Because most microbiological testing only take a portion of thehomogenate (e.g., 0.1 mL or 1 mL for plating; 2.5 μL for direct PCR),the homogenization by dilution method decreases the detectability of theorganisms of interest by as much as 1,500,000 times. To increase thedetectability, a lengthy enrichment procedure is incorporated to allow asingle viable cell to proliferate to millions so that it can bedetected. This procedure costs 24 to 48 hours delay in obtainingdetection data and more days in decision making time due to subsequentconfirmation steps. It is also important that the samples be preservedproperly and extraction be done as close to sampling time as possible.If the sample has changed before extraction, the results do notrepresent the tested lot.

For most conventional sampling programs, the percentage of negativesamples exceeds 99%. Based on simple modeling, one can easily concludethat the number of cells of the organism of interest is seldom more than1 and probably rarely more than 5 based on a Poisson distribution. Withso few cells present, the enrichment is often done in the extractionbuffer. This is critical with the small sampling but does not make thistype of testing any more representative of the lot under test.

However, this conventional enrichment and detection can be applied toextract from an aggregating sampler when presence or absence of thetarget organism is the desired goal. Modes of detection are consideredherein.

When extracting the sampling materials these same considerations apply.However, the impact on the results of failing to extract one organism isan order of magnitude or smaller due to the larger effective sample. Inaddition, the cassettes afford the opportunity to begin the extractionfaster by adding the fluid immediately after sampling providing maximaltime for the extraction to occur while avoiding human interaction.

When extracting a 24×8 inch sampling sheet of a poly-olefin sampler, avolume of 100 to 200 mls of extraction solution is generallyappropriate. The composition of this solution is driven by the planneddetection system and is generally defined in the associated method.

Examples of Concentration

During the concentration, two classes of materials may need to beremoved, the small, <100,000 molecular weight, and the very large, >50microns. In addition, the sample may need to be concentrated to about 1mL to be compatible with the screening detection system. Given that theextraction generally starts at about 200 mL, there is a large amount ofwater to remove.

Two schemes are practical. Traditionally, one can filter the extractionfluid through an inert 50 microns cut off filter and then sediment theorganisms of interest by centrifugation. The resulting pellets can bere-suspended in an appropriate buffer and taken on to cleaning.Alternatively, after filtration, the small molecules and water can beremoved osmotically with adsorbents or pressure and a semi-permeablemembrane such as used for reverse osmosis or ultrafiltration. The latteris more amenable to automation as the resultant concentrated sampleremains in solution. However, this option may require a for purposemodule to be executed.

It is important to maintain the connection to the initial Meta datathrough this process. If enough of interfering material is removed andthe sample is sufficiently concentrated, the cleaning step examined nextcan be skipped and moving directly to the screening determination. Thisdecision determination may be made of a case by case basis.

It is also possible to use non-specific binding such as a cationexchange surface to collect that target organisms and remove them fromthe bulk solution. Such as an approach would partially combineconcentration and cleaning. This approach is most practical when thethere are few larger particles to interfere.

Examples of Cleaning

At this point in the process, samples have been greatly reduced involume but the organisms of interest have not been segregated from otherorganism so the signal to noise ratio is till problematic. In addition,further concentration may be needed for detection without enrichmentduring which one organism is converted to many at the cost of time anddelay in decision making.

Several schemes are practical but all involve binding the organisms ofinterest in a small area such as a microfluidized or nanofluidizedchannel. This channel or area may or may not be filled with surfaceactivated nanofiber. Utilizing elasto-inertial microfluidics, theviscoelastic flow enables size based migration of larger particles intoa non-Newtonian solution, while smaller bacteria remain in thestreamline of the blood sample entrance and can be separated. It istempting to consider surface activated magnetic particles; however, themechanical manipulation of these particles to achieve the desired smallvolume is a larger engineering challenge than activating the smallsurface area. However, any binding geometry that fixes the organisms ofinterest and any other organisms selected to represent the othermicroflora in an appropriate small volume can be used.

The surface activation may require numerous active binding sites inclose proximity. The mixed DNA primer arrays of SnapDNA are one class ofmaterials. Another class is a cocktail of antibodies for all theorganisms of interest.

The binding mechanism may bind all the organism of interest. Theseorganisms may be sufficiently bound that other organisms and materialsare selectively removed from the area of binding as clean fluid ispassed through the channel.

The motive force to move the cells through the channel can be themechanical action of fluid flow, electrostatic as the surface of mostbacteria is negative, or a size pumping action such as practiced withferromagnetic particles.

Examples of Screening Detection

With the partially purified organism or organisms of interest bound in asmall area or small volume if the cleaning step proves unnecessary, manyapproaches are available for screening detection for process control.The functional requirements are clearer. First and foremost is thatenrichment culture takes substantial amounts of time, and such should beeliminated or reduced in one or more cases. Second, the screening metricmay need to be an index suitable for statistical process control. Thisimplies a measurement with many states as opposed to binary 0 and 1. Themagnitude of this metric may relate to the extent of deviation fromnormal operation and therefore the likelihood that an outbreak couldoccur. Under these conditions, the detection of deviation can be basedon the classic rules for control charting. Furthermore, trend detectingrules have the potential to detect problems before significantdeviations have occurred. The statistically based Westcard rules provideone basis for trend analysis.

Expressed differently, an index may be needed that tallies the relativepresence of beneficial organisms and potential pathogens and aggregatesthis information in a useful way. As more information is acquired aboutspecific products, the power of big data will come into play. However,at the simplest levels, the aggregate level of potential pathogen is auseful screening tool. The relative balance between potential pathogensand beneficial organisms is a more sophisticated analysis to compensatefor seasonal variations that are inherent in many products. It isreasonable to expect to develop indices that are product specific.

The information behind indices is evolving rapidly. The simplest usefulindex is a ratio of pathogen intensity to a benign organism. These canbe generated by many means including classical enumeration with plating,but the classical methods are two slow to meet the functionalrequirements outlined above. However, given the concentration of theorganisms on the cleaning substrate or the concentrated extraction, itis possible to go directly to qPCR in some cases to generate index data.

There are two schemes for generating this type of data at its mostsophisticated level. First, one can use a collection of ligands(antibodies, aptamers, or others) that bind and tag all the organisms ofpotential interest yielding a collection of signals that are multiplexedinto a family of useful channels. Alternatively, one can generate anarray of specific binding interactions that are analyzed chemometricallyto yield a metric. The latter approach will be faster and probably lessexpensive after the research and analysis is done.

As an example of the first scheme would be a using a mixture ofconjugated antibodies to bind to all types of cells of interest. Forproduce, hemolytic E. coli, Salmonella and Listeria are of greatestinterest. Poultry focuses on Campylobacter and Salmonella. Otherindustries have other and additional interests. These antibodies can bebound to the organisms bound to the cleaning substrate. The retainedtags with either a fluorescent probe or an enzyme provide signalamplification and detection. When the cleaning region as a small enoughcross section, the signal from the hundreds to thousands of cells on thecleaning substrate are detectable. Micro and nano fluidization arenecessary. However, specific detection protocols can be evaluated at amacro scale using an appropriately instrumented microscope that can beused to measure the signal from a small area where cells have beencollected. For speed to market, multiple detectors can be run inparallel or series to generate similar multiple channels of data.

The alternative scheme invokes the lab on a chip concept. By building anarray of binding sites, the composition of the samples can be queried.PCR can amplify the contents selectively with a collection of primers.Such detectors can evolve to providing both the screening detection ofthis step and the ultimate secondary screening. However, it is likely toremain a two-step process due to the economics.

A number of technologies can meet these requirements including but notlimited to, for example: 1) Sensors where the vibrational frequencies(which may for example include optical waveguide), impedance, or otherproperties of a transistor are modified by the binding of the organismsof interest to the sensor. This approach may require that the sensor bebuilt into the surface of the channel. Another technology may include,2) qPCR where the cells are laid in place and the number of copies ofthe organisms of interest, or the number of ribosomes of the organismsof interest are estimated. Another technology may include, 3) Use taggedantibodies to light up the organisms of interest so that they candetected on the absorbent surface spectrophotometrically. Enzyme,fluorescent probes, and other materials to amplify the signal can beused.

It may be desirable to avoid confirmation of the presence of one or morespecific pathogens during this screening. This is where the tradebetween LOD and speed to useful information. If the signal is not aboutthree times background, no further action is warranted.

Given that economics may play a role in testing, it should be noted thatthe sampling approach is compatible with various compositing strategies.Samples can be composited anywhere along the processing path beforedetection. In various situations, one position will be more advantageousthan others. Pooling samples prior to extraction reduces work butreduces the resolution of the results if a positive result is found.However, if the result is negative tremendous savings are achieved.After concentrating and cleaning would allow sampled to be retested ifonly a portion of the extracted sample is used in a wet pooling approachwhich can avoid detections costs. Each system needs to be evaluated todetermine how to best control costs.

Examples of Confirmation

The confirmation step will be applied when there is reason to suspectthat a pathogen is present. The technology in this area is evolvingrapidly with many new approaches and efficiencies being developed andintroduced. Some will use the techniques suggested for screening withmore specific reagents for the confirmation. At present, all the commonprocedures rely on either molecular biology or ligand binding reactions.Various strategies have been developed to amplify the signal noise ratioand to identify the contaminant to the desired specificity. The desiredspecificity ranges from simple speciation to identify specificserotypes.

For confirmation, a concentrated sample such as afforded the screeningdetection above may be included. The concentration may be included dueto the small volumes that are compatible with these types of procedures.The organisms, the surface antigens from the organisms, or the nucleicacid from the organisms may need to be extracted from the screeningsystem to the extent that these materials can interact with the reagentsof the confirmation procedure. In other words, there is not a prioryreason that the detection module could not be engineered for a secondround of chemistries. Any one of these materials may contain theinformation necessary to characterize the contaminant. There are anumber of these processes and they are being improved. The choice ofapproach will be driven by cost, desired specificity and the desiredspeed.

Many strategies are available to amplify the base signal from thesematerials that will still be present at only modest concentration in thetypical sample. The yield of material from the hundreds to thousands oforganisms of interest bound to the screening detector platform is stillvery small. Amplification based on radio isotopes are largely out offavor but still possible. However, enzyme systems are still in use andnew enzymes strategies are still being developed such as those used forELISA methods or with a luciferase. Fluorescent tags on specificantibodies such as those proposed for the screening determination areless useful for this purpose due the high potential for crossreactivity. However, antibodies of this type are the basis for theserotyping classifications that up until recently has been the standardfor characterization. These older serotyping assays may requireisolation and growing the organisms.

Increasingly characterization is based on the presence of sequences ofnucleic acid. These can be nuclear DNA, ribosomal RNA, or messenger RNA.At the extreme, it is now practical to sequence the entire genome of theorganism. Increasing specificity is useful for identification of thesource a contamination. However, it also presents a potential liabilityin the case of an outbreak of illness.

The molecular assays are built around the use of enzymes to replicatesequences of nucleic acid to generate a large enough signal fordetection. Various primers are used to select which portions are copied;up to and including the entire genome. For measurement, variousfluorescent tags are used. One of the newest techniques utilizes themelting and binding of target material to known sequences to generate acomplex matrix of information that can be used chemometrically in lieuof complete sequence data.

Examples of Reporting and Roll Up

Both the screening detection and the confirmation results are reporteddirectly to a sequel database. This reporting may not require humanintervention if the included quality assurance standards (positive andnegative controls) fall within normal ranges. This avoids transcriptionand transposition errors. Digital records are more reliable and accuratethan manual records.

Both detectors should be part of the “Internet of Things”. Thisconnectivity allows results to be pushed to operators on the floorallowing for the rapid release of product or for the redisposition ofproduct if a potential issue is identified that may need to beaddressed. Once the results are in an appropriate database, varioususers can have customized interfaces providing the information. Some mayneed to track individual results. Others may be more interested intrends and averages. There may be provided a class of users that maywant to aggregate even larger data sets to compare across locations.

There are many platforms available for extracting information from thedatabase. For example, but not limited thereto, Ignition has provenuseful in this record as it is open source allowing customization.

In accordance with an aspect of the disclosure, a method for microbialsampling food may include gathering a microbial sampling from one ormore food items, extracting microorganisms from the microbial sampling,concentrating the microorganisms, cleaning the microorganisms, tallyinga relative presence of the microorganisms and any potential pathogens,aggregating information of a microorganism tally from the tallying ofmicroorganisms into a microorganism report, confirming the microorganismtally, and reporting the microorganism report of the microorganismtally.

In some cases, gathering the microbial sampling from the one or morefood items includes sampling, using an aggregating sampler, the one ormore food items that include a production lot of produce or meatcreating one or more samples that makes up the microbial sampling. Theone or more samples may be configured to be processed to indicate ifpathogens are present at no greater than a normal background.

In some cases, the method may further include assessing, using anaggregating sampler, a level of cross contamination control to validateor verify a wash process. Gathering the microbial sampling from the oneor more food items may include providing an aggregating sampler at asampling location. The sampling location may be at least one of in afield, at harvest, just after dumping or cutting, in a wash system, orafter the wash system.

In some cases extracting includes enriching the microbial sampling, andadding fluid to the microbial sample. In some cases concentratingincludes filtering extraction fluid of the microbial sampling using atleast one of centrifugation filtering or osmotically filtering. In somecases, cleaning includes binding the microorganisms in a small areaincluding one or more of a microfluidized or nanofluidized channel. Insome cases tallying includes using a collection of ligands that bind andtag all the microorganisms of potential interest yielding a collectionof signals that are multiplexed into a family of useful channels. Insome cases ligands include one or more of antibodies, primers, andaptamers.

In some cases tallying includes generating an array of specific bindinginteractions that are analyzed chemometrically to yield a metric. Themethod may further include building an array of binding sites, whereinthe composition of the samples can be queried, and amplifying, using aPCR, the contents selectively with a collection of primers.

In some cases confirming includes extracting surface antigens from theorganisms or nucleic acid from the organisms from a screening system tothe extent that these materials can interact with the reagents of aconfirmation procedure. In some cases confirming further includesamplifying a base signal of the microorganism tally.

In some cases, the method may further include use of an index as asurrogate for direct results regarding presence or absence of organismsof interest. In some cases, the method may further include use of astatistical process control for detecting deviations in microbial flora.

In accordance with an aspect of the disclosure, a method of applyingaggregating sampling to food items including providing at least oneaggregating sampler at one or more sampling locations, and sampling,using the at least one aggregating sampler, a production lot of produceor meat creating one or more samples that makes up a microbial sampling.

In some cases the one or more samples are configured to be processed toindicate if pathogens are present at no greater than a normalbackground. In some cases, the one or more sampling locations includesat least one of in a field, at harvest, just after dumping or cutting,in a wash system, or after the wash system. The method may furtherinclude assessing, using the aggregating sampler, a level of crosscontamination control to validate or verify a wash process.

In accordance with an aspect of the disclosure, an apparatus formicrobial sampling, including means for gathering a microbial samplingfrom one or more food items, means for extracting microorganisms fromthe microbial sampling, and means for concentrating the microorganisms,means for cleaning the microorganisms, means for tallying a relativepresence of the microorganisms and any potential pathogens, means foraggregating information of the microorganism tally into a microorganismreport, means for confirming the microorganism tally, and means forreporting the microorganism report of the microorganism tally.

In accordance with an aspect of the disclosure, an apparatus formicrobial sampling, includes at least one processor configured togenerate control signals for controlling gathering a microbial samplingfrom one or more food items, extracting microorganisms from themicrobial sampling, concentrating the microorganisms, cleaning themicroorganisms, tallying a relative presence of the microorganisms andany potential pathogens, aggregating information of the microorganismtally into a microorganism report, and confirming the microorganismtally, and a transmitter configured to transmit the microorganism reportof the microorganism tally. In some cases the apparatus may furtherinclude an aggregating sampler configured to gather the microbialsampling.

In accordance with an aspect of the disclosure, a non-transitorycomputer readable medium for microbial sampling having instructionsstored thereon for gathering a microbial sampling from one or more fooditems, extracting microorganisms from the microbial sampling,concentrating the microorganisms, cleaning the microorganisms, tallyinga relative presence of the microorganisms and any potential pathogens,aggregating information of the microorganism tally into a microorganismreport, confirming the microorganism tally, and reporting themicroorganism report of the microorganism tally.

In accordance with an aspect of the disclosure, a method for samplingfood including concentrating microorganisms and removing interference,tallying a relative presence of the microorganisms and any potentialpathogens, and aggregating information of the microorganism tally into amicroorganism report. In accordance with an aspect of the disclosure, asystem capable of implementing one or more of the novel aspectsdiscussed in this application disclosure.

In accordance with an aspect of the disclosure, a microbial aggregatingsampler, including a covering including a microbial sampling materialwith a pocket formed in the covering to receive an appendage or a toolfor handling of the covering.

In some cases, the covering includes an attachment feature formed in thepocket to receive the tool. In some cases, the attachment featureincludes one of a hole formed through the covering, a loop positionedwithin the pocket to receive an end of the tool there through, and a tabpositioned within the pocket for an end of the tool to attach thereto.

In some cases the covering includes a sheath formed in the pocket toreceive a digit of an appendage. In some cases the pocket is formedthrough the covering such that the appendage or the tool for handlingthe covering extends through the covering. In some cases the covering iscompletely formed from the microbial sampling material. In some casesthe covering includes two sheets attached to each other to form thepocket. In some cases the covering includes a single sheet folded andattached to itself to form the pocket.

Other Applications Beyond Bacterial Testing

The aggregating sampler can be used to sample for additional analytesbeyond bacteria including yeast, molds, viruses, allergens, toxins suchas aflatoxin, or particulates such as dust. The commonality is thesurface presence of the analyte at low levels that can be concentrated.The basic process is the same with the same key steps. The biggestdifferences will be in detection strategy, but these strategies are wellknown by those who study these analytes.

The methods disclosed herein comprise one or more steps or actions forachieving the described method. The method steps and/or actions may beinterchanged with one another without departing from the scope of theclaims. In other words, unless a specific order of steps or actions isspecified, the order and/or use of specific steps and/or actions may bemodified without departing from the scope of the claims.

As used herein, a phrase referring to “at least one of a list of” itemsrefers to any combination of those items, including single members. Asan example, “at least one of: a, b, or c” is intended to cover a, b, c,a-b, a-c, b-c, and a-b-c, as well as any combination with multiples ofthe same element (e.g., a-a, a-a-a, a-a-b, a-a-c, a-b-b, a-c-c, b-b,b-b-b, b-b-c, c-c, and c-c-c or any other ordering of a, b, and c). Asused herein, including in the claims, the term “and/or,” when used in alist of two or more items, means that any one of the listed items can beemployed by itself or any combination of two or more of the listed itemscan be employed. For example, if a composition is described ascontaining components A, B, and/or C, the composition can contain Aalone; B alone; C alone; A and B in combination; A and C in combination;B and C in combination; or A, B, and C in combination.

As used herein, the term “determining” encompasses a wide variety ofactions. For example, “determining” may include calculating, computing,processing, deriving, investigating, looking up (e.g., looking up in atable, a database or another data structure), ascertaining and the like.Also, “determining” may include receiving (e.g., receiving information),accessing (e.g., accessing data in a memory) and the like. Also,“determining” may include resolving, selecting, choosing, establishingand the like.

The previous description is provided to enable any person skilled in theart to practice the various aspects described herein. Variousmodifications to these aspects will be readily apparent to those skilledin the art, and the generic principles defined herein may be applied toother aspects. Thus, the claims are not intended to be limited to theaspects shown herein, but is to be accorded the full scope consistentwith the language claims, wherein reference to an element in thesingular is not intended to mean “one and only one” unless specificallyso stated, but rather “one or more.” For example, the articles “a” and“an” as used in this application and the appended claims shouldgenerally be construed to mean “one or more” unless specified otherwiseor clear from the context to be directed to a singular form. Unlessspecifically stated otherwise, the term “some” refers to one or more.Moreover, the term “or” is intended to mean an inclusive “or” ratherthan an exclusive “or.” That is, unless specified otherwise or clearfrom the context, the phrase, for example, “X employs A or B” isintended to mean any of the natural inclusive permutations. That is, forexample the phrase “X employs A or B” is satisfied by any of thefollowing instances: X employs A; X employs B; or X employs both A andB. All structural and functional equivalents to the elements of thevarious aspects described throughout this disclosure that are known orlater come to be known to those of ordinary skill in the art areexpressly incorporated herein by reference and are intended to beencompassed by the claims. Moreover, nothing disclosed herein isintended to be dedicated to the public regardless of whether suchdisclosure is explicitly recited in the claims. No claim element is tobe construed under the provisions of 35 U.S.C. § 112, sixth paragraph,unless the element is expressly recited using the phrase “means for” or,in the case of a method claim, the element is recited using the phrase“step for.”

The various operations of methods described above may be performed byany suitable means capable of performing the corresponding functions.The means may include various hardware and/or software component(s)and/or module(s), including, but not limited to a circuit, anapplication specific integrated circuit (ASIC), or processor. Generally,where there are operations illustrated in figures, those operations mayhave corresponding counterpart means-plus-function components withsimilar numbering.

The various illustrative logical blocks, modules and circuits describedin connection with the present disclosure may be implemented orperformed with a general purpose processor, a digital signal processor(DSP), an application specific integrated circuit (ASIC), a fieldprogrammable gate array (FPGA) or other programmable logic device (PLD),discrete gate or transistor logic, discrete hardware components, or anycombination thereof designed to perform the functions described herein.A general-purpose processor may be a microprocessor, but in thealternative, the processor may be any commercially available processor,controller, microcontroller, or state machine. A processor may also beimplemented as a combination of computing devices, e.g., a combinationof a DSP and a microprocessor, a plurality of microprocessors, one ormore microprocessors in conjunction with a DSP core, or any other suchconfiguration.

If implemented in hardware, an example hardware configuration maycomprise a processing system in a wireless node. The processing systemmay be implemented with bus architecture. The bus may include any numberof interconnecting buses and bridges depending on the specificapplication of the processing system and the overall design constraints.The bus may link together various circuits including a processor,machine-readable media, and a bus interface. The bus interface may beused to connect a network adapter, among other things, to the processingsystem via the bus. The network adapter may be used to implement thesignal processing functions of the PHY layer. In the case of a userterminal 120 (see FIG. 1); a user interface (e.g., keypad, display,mouse, joystick, etc.) may also be connected to the bus. The bus mayalso link various other circuits such as timing sources, peripherals,voltage regulators, power management circuits, and the like, which arewell known in the art, and therefore, will not be described any further.The processor may be implemented with one or more general-purpose and/orspecial-purpose processors. Examples include microprocessors,microcontrollers, DSP processors, and other circuitry that can executesoftware. Those skilled in the art will recognize how best to implementthe described functionality for the processing system depending on theparticular application and the overall design constraints imposed on theoverall system.

If implemented in software, the functions may be stored or transmittedover as one or more instructions or code on a computer readable medium.Software shall be construed broadly to mean instructions, data, or anycombination thereof, whether referred to as software, firmware,middleware, microcode, hardware description language, or otherwise.Computer-readable media include both computer storage media andcommunication media including any medium that facilitates transfer of acomputer program from one place to another. The processor may beresponsible for managing the bus and general processing, including theexecution of software modules stored on the machine-readable storagemedia. A computer-readable storage medium may be coupled to a processorsuch that the processor can read information from, and write informationto, the storage medium. In the alternative, the storage medium may beintegral to the processor. By way of example, the machine-readable mediamay include a transmission line, a carrier wave modulated by data,and/or a computer readable storage medium with instructions storedthereon separate from the wireless node, all of which may be accessed bythe processor through the bus interface. Alternatively, or in addition,the machine-readable media, or any portion thereof, may be integratedinto the processor, such as the case may be with cache and/or generalregister files. Examples of machine-readable storage media may include,by way of example, RAM (Random Access Memory), flash memory, phasechange memory, ROM (Read Only Memory), PROM (Programmable Read-OnlyMemory), EPROM (Erasable Programmable Read-Only Memory), EEPROM(Electrically Erasable Programmable Read-Only Memory), registers,magnetic disks, optical disks, hard drives, or any other suitablestorage medium, or any combination thereof. The machine-readable mediamay be embodied in a computer-program product.

A software module may comprise a single instruction, or manyinstructions, and may be distributed over several different codesegments, among different programs, and across multiple storage media.The computer-readable media may comprise a number of software modules.The software modules include instructions that, when executed by anapparatus such as a processor, cause the processing system to performvarious functions. The software modules may include a transmissionmodule and a receiving module. Each software module may reside in asingle storage device or be distributed across multiple storage devices.By way of example, a software module may be loaded into RAM from a harddrive when a triggering event occurs. During execution of the softwaremodule, the processor may load some of the instructions into cache toincrease access speed. One or more cache lines may then be loaded into ageneral register file for execution by the processor. When referring tothe functionality of a software module below, it will be understood thatsuch functionality is implemented by the processor when executinginstructions from that software module.

Also, any connection is properly termed a computer-readable medium. Forexample, if the software is transmitted from a website, server, or otherremote source using a coaxial cable, fiber optic cable, twisted pair,digital subscriber line (DSL), or wireless technologies such as infrared(IR), radio, and microwave, then the coaxial cable, fiber optic cable,twisted pair, DSL, or wireless technologies such as infrared, radio, andmicrowave are included in the definition of medium. Disk and disc, asused herein, include compact disc (CD), laser disc, optical disc,digital versatile disc (DVD), floppy disk, and Blu-ray® disc where disksusually reproduce data magnetically, while discs reproduce dataoptically with lasers. Thus, in some aspects computer-readable media maycomprise non-transitory computer-readable media (e.g., tangible media).In addition, for other aspects computer-readable media may comprisetransitory computer-readable media (e.g., a signal). Combinations of theabove should also be included within the scope of computer-readablemedia.

Thus, certain aspects may comprise a computer program product forperforming the operations presented herein. For example, such a computerprogram product may comprise a computer-readable medium havinginstructions stored (and/or encoded) thereon, the instructions beingexecutable by one or more processors to perform the operations describedherein. For example, instructions for performing the operationsdescribed herein and illustrated in the appended figures.

Further, it should be appreciated that modules and/or other appropriatemeans for performing the methods and techniques described herein can bedownloaded and/or otherwise obtained by a user terminal and/or basestation as applicable. For example, such a device can be coupled to aserver to facilitate the transfer of means for performing the methodsdescribed herein. Alternatively, various methods described herein can beprovided via storage means (e.g., RAM, ROM, a physical storage mediumsuch as a compact disc (CD) or floppy disk, etc.), such that a userterminal and/or base station can obtain the various methods uponcoupling or providing the storage means to the device. Moreover, anyother suitable technique for providing the methods and techniquesdescribed herein to a device can be utilized.

It is to be understood that the claims are not limited to the preciseconfiguration and components illustrated above. Various modifications,changes and variations may be made in the arrangement, operation anddetails of the methods and apparatus described above without departingfrom the scope of the claims.

What is claimed is:
 1. A method for microbial sampling food comprising:gathering a microbial sampling from one or more food items with amicrobial sampling sheet, the microbial sampling sheet comprising anon-woven synthetic cloth; extracting microorganisms from the microbialsampling, wherein the microorganisms include non-pathogenicmicroorganisms and any potential pathogens if present; concentrating themicroorganisms; cleaning the microorganisms; determining an amount ofthe microorganisms and determining a presence of any potentialpathogens; aggregating information from the determining of the amount ofthe microorganisms into a microorganism report; confirming thedetermination of the amount of the microorganisms; and reporting themicroorganism report of the determination of the amount of themicroorganisms and the presence of any potential pathogens.
 2. Themethod of claim 1, wherein gathering the microbial sampling from the oneor more food items comprises: sampling, using an aggregating sampler,multiple food items within a production lot of produce or meat creatingone or more aggregate samples that makes up the microbial sampling bycontacting an outer sampling surface of the microbial sampling sheet tothe one or more food items to be sampled.
 3. The method of claim 1,wherein the one or more food items comprises meat.
 4. The method ofclaim 1, wherein the microbial sampling sheet comprises a sampling sheetof non-woven polypropylene fabric.
 5. The method of claim 4, wherein thesampling sheet is 24×8 inches.
 6. The method of claim 1, whereingathering the microbial sampling comprises manual sampling with thesampling sheet.
 7. The method of claim 1, further comprising: assessing,using an aggregating sampler, a level of cross contamination control tovalidate or verify a wash process.
 8. The method of claim 1, whereingathering the microbial sampling from the one or more food itemscomprises: providing an aggregating sampler and/or manual sampling withthe microbial sampling sheet at a sampling location, wherein thesampling location is at least one of in a field, at harvest, just afterdumping or cutting, in a wash system, or after the wash system.
 9. Themethod of claim 1, further comprising: placing the sampling sheet aftersampling within a bag for stomaching and/or enriching within the bag.10. The method of claim 1, wherein concentrating comprises: filteringextraction fluid of the microbial sampling using at least one ofcentrifugation filtering or osmotically filtering.
 11. The method ofclaim 1, wherein cleaning comprises: binding the microorganisms in asmall area including one or more of a microfluidized or nanofluidizedchannel.
 12. The method of claim 1, wherein determining the amount ofthe microorganisms comprises: using a collection of ligands that bindand tag all microorganisms of potential interest yielding a collectionof signals that are multiplexed into a family of useful channels,wherein ligands include one or more of antibodies, primers, andaptamers.
 13. The method of claim 1, wherein determining the amount ofthe microorganisms comprises: generating an array of specific bindinginteractions that are analyzed chemometrically to yield a metriccomprising: building an array of binding sites, wherein a composition ofthe samples can be queried; and amplifying, using a PCR, the samplesselectively with a collection of primers.
 14. The method of claim 1,wherein confirming comprises: extracting surface antigens from themicroorganisms or nucleic acid from the microorganisms from a screeningsystem to an extent that these materials can interact with reagents of aconfirmation procedure; and amplifying a base signal of the determiningthe amount of the microorganisms.
 15. The method of claim 1, furtherincluding use of one or more of an index as a surrogate for directresults regarding presence or absence of organisms of interest, or astatistical process control for detecting deviations in microbial flora.16. A method of applying aggregating sampling to food items, the methodcomprising: providing at least one aggregating sampler at one or moresampling locations, wherein the aggregating sampler comprises amicrobial sampling sheet having an outer sampling surface for contactingthe food items to be sampled during sampling with a tool, appendage,apparatus or by manual sampling, wherein the microbial sampling sheetcomprises a non-woven synthetic cloth; and sampling, using the at leastone aggregating sampler, multiple food items within a production lot ofproduce or meat by contacting the multiple food items with theaggregating sampler to obtain one or more aggregate samples that makesup a microbial sampling.
 17. The method of claim 16, further comprising:determining an amount of the microorganisms from the one or moreaggregate samples; and indicating if pathogens are present at no greaterthan a normal background based on the determination of the amount of themicroorganisms.
 18. The method of claim 16, wherein the one or moresampling locations includes at least one of in a field, at harvest, justafter dumping or cutting, in a wash system, or after the wash system.19. The method of claim 16, further comprising: assessing, using theaggregating sampler, a level of cross contamination control to validateor verify a wash process.
 20. The method of claim 17, wherein the amountof the microorganisms is determined from the one or more aggregatesamples without enrichment.
 21. The method of claim 16, wherein the fooditems comprises meat.
 22. The method of claim 16, wherein the microbialsampling sheet comprises a sheet of non-woven polypropylene fabric. 23.The method of claim 16, wherein the sampling sheet is 24×8 inches. 24.The method of claim 16, wherein sampling comprises manual sampling withthe microbial sampling sheet.
 25. The method of claim 16, furthercomprising: placing the sampling sheet after sampling within a bag forstomaching and/or enriching within the bag.